The ancestors of modern organisms were colonized by viruses that were very rarely beneficial, and often deleterious to the host. Inevitably, hosts adapted to limit viral replication, and in so doing evolved various autonomous antiviral defense mechanisms. One of these is TRIM5, which is multimeric protein that recognizes incoming retroviral capsids, but its precise mechanisms of action are unclear. In specific aim 1 we will develop new techniques to understand what happens to various incoming components of retroviral particles as they complete the early steps of their life cycle. These assays will very likely illuminate the mechanisms by which TRIM5 proteins and other antiretroviral factors exert their inhibitory effects. Moreover, these techniques are likely to have a wide range of applications in the study of these steps of the retroviral life cycle. In specific aim 2 we will derive TRIM5-resistant HIV-1 capsids that will identify residues on the surface of the HIV-1 capsid that are targeted by TRIM5 and will serve a useful tools to probe the mechanism of action of TRIM5. Additionally these capsids will be used for generating simian-tropic HIV- strains that can replicate in rhesus macaques. In addition to known antiretroviral proteins like TRIM5, there are several reasons to think that there are more, perhaps many more, antiviral host defense genes in to be discovered. In specific aim 3, we will attempt to identify such genes by constructing focused, arrayed cDNA libraries, each consisting of tens to hundreds of candidate genes, that either (i) have key properties exhibited by known antiretroviral genes or (ii) are selectively expressed in cells that exhibit constitutive or induced resistance to HIV-1 or SIV infection. These genes libraries will be arrayed each gene will be individually tested for its ability to inhibit retrovirus replication.